PROJECT SUMMARY Deregulated Wnt/?-catenin signaling is a common feature of colorectal cancer (CRC), but how this pathway corrupts target gene expression is not fully understood. The T-cell factor/Lymphoid enhancer factor (TCF/Lef; hereafter TCF) transcription factors bind Wnt-responsive DNA elements (WREs) to control Wnt/?-catenin target gene expression. Of the four TCF family members (TCF7, LEF1, TCF7L1, and TCF7L2) very little is known about TCF7L1 in CRC and the set of target genes it directly regulates. By using shRNAs to deplete TCF7L1 in established human CRC lines, we found that it promotes cell growth. Although microarray analysis of transcripts differentially expressed in TCF7L1 knockdown cells versus controls uncovered hundreds of genes, surprisingly, we failed to identify a Wnt target gene signature among this list. Moreover, published ChIP-Seq data indicates that a substantial fraction of TCF-bound DNA elements lack consensus TCF binding motifs. These findings suggest that TCF7L1 may primarily function outside the canonical Wnt signaling pathway to promote oncogenesis. Additional work is needed to identify the constellation of direct target genes that TCF7L1 regulates and whether its DNA-binding function is required to promote CRC growth. In Aim 1, we will identify the TCF7L1-transcriptome by conducting unbiased and genome-wide functional genomics approaches in control and TCF7L1-depleted CRC cells. In TCF7L1-depleted lines, we will introduce wild-type and DNA-binding deficient TCF7L1 cDNAs to classify targets whose expression is regulated independently of direct TCF7L1 binding to DNA. In Aim 2, we will determine whether the DNA-binding capacity of TCF7L1 is required for CRC cell proliferation, progression through the cell cycle, growth in an anchorage-independent manner, and tumorigenesis in vivo. We will also assess whether non-canonical targets are differentially expressed in primary human colonic tissues and tumors. The vast majority of existing therapeutics designed to target the Wnt pathway have focused on nuclear TCF/?-catenin complexes as the key regulator of the CRC transcriptome. Our findings will establish TCF7L1 as a critical regulator of genes outside the canonical Wnt/?- catenin pathway and will open a whole new field of research to exploit those targets for therapeutic intervention.